The goals of this project are to investigate the role of altered ion homeostasis in cell injury and protective adaptation. We previously demonstrated that an increase in cytosolic free [Ca2+] (Cai) is causally involved in cell injury, and that reducing the rise in Cai is protective. This project has two lines of investigation. One series of studies are investigating the mechanisms involved in protective adaptation (preconditioning). Brief intermittent periods of stress (ischemia, H2O2, osmotic stress etc.), termed preconditioning, provide protection against injury during a subsequent longer period of stress. Studies have shown that preconditioning reduces the rise in Cai. Current studies involve elucidating the signaling pathways that are responsible for this reduced rise in Cai. We have shown that activators of PKC mimic preconditioning and that inhibitors of PKC block preconditioning. We also find that PC causes an increase in 12-lipoxygenase metabolites and that addition of a 12-lipoxygenase metabolite mimics preconditioning and inhibitors of 12-lipoxygenase metabolism block preconditioning. In an attempt to order PKC and 12-lipoxygenase metabolites in preconditioning, we find that PKC activators increase 12-lipoxygenase metabolism and inhibitors of PKC block the preconditioning induced increase in 12-lipoxygenase metabolism. We are also investigating the downstream targets of 12-lipoxygenase metabolism. A mitochondrial K-channel is reported to be involved in preconditioning, and 5 HD, a putative specific inhibitor of the mitochondrial K-ATP channel has been reported to block preconditioning. We find that 5HD blocks the protective effects of 12-HpETE (the 12-lipoxygenase metabolite that mimics preconditioning). A second series of studies are investigating the basis of gender differences in the response to cell injury. Studies using mice that overexpress the plasma membrane Na/Ca exchanger have shown that perfused hearts from male, but not female Na/Ca overexpressor mice have poor recovery of function following ischemia, most likely due to an increase in Cai, because a larger proportion of the increased intracellular [Na+] which occurs during ischemia exits the cell in exchange for Ca2+. The functional recovery of hearts from bilateral ovariectomized female transgenic mice was significantly worse than that of non-ovariectomized female transgenics and similar to that of male transgenic mice, suggesting that estrogen counters the effects of overexpression of the Na/Ca exchanger. Interestingly we also find that in mice that overexpress the ?-adrenergic receptor, there is increased injury in male vs. female ischemic hearts. We are investigating mechanisms responsible for these gender based differences in cell injury.